Influenza virus is a major public health problem in the United States and worldwide. To better understand the cellular events that occur during influenza virus infection, we have been studying the shut-off of host cell protein synthesis and the selective translation of viral mRNAs in influenza virus-infected cells. We have accumulated evidence that the molecular strategies employed by influenza virus to accomplish these goals are intimately intermeshed with the host cell defense and stress-response pathways. In Specific Aim 1, we will further define the mechanisms of selective translation. We have identified a cellular RNA-binding protein, GRSF-1, that binds to the 5' untranslated region (UTR) of influenza virus mRNAs. This is significant, since we earlier demonstrated that the 5' UTR was both necessary and sufficient to redirect the host cell protein synthesizing machinery to translate only viral mRNAs. We hypothesize that GRSF-1 interacts with the 5' UTR of influenza virus mRNAs to upregulate viral protein synthesis. To test this hypothesis, we will perform separate in vitro functional assays and in vivo experiments with wild-type and transdominant mutant GRSF-1. Specific Aim 2 focuses on the stress-response pathway activated by influenza virus to ensure efficient viral mRNA translation. Influenza virus recruits the cellular TPR protein, P58IPK, PK, to down-regulate the interferon-induced PKR protein kinase, thereby keeping protein synthetic activity high in a virus-infected cell. In the absence of this regulation, activation of PKR by viral RNAs results in the phosphorylation of the eukaryotic initiation factor, eIF-2alpha and inhibition of protein synthesis initiation. In this aim, we will dissect the P58IPK/PKR pathway and examine the roles played by the P58IPK regulators, hsp40, P52rIPK, and the molecular chaperone hsp70, which we now hypothesize plays a key role in the downregulation of PKR. Finally, in Aim 3, we propose to construct a knockout mouse with a deletion of the P58IPK gene. We will examine the effects of deleting P58IPK on viral and cellular mRNA translation and gene expression, both in the null mice and in fibroblasts prepared from these mice. Together, the studies outlined will contribute to a better understanding of eukaryotic protein synthesis regulation, which may ultimately provide insights into novel antiviral therapeutics.